We will develop new technologies to employ quantum dots in biological imaging in vitro. In an evolving program, quantum dot technology will be developed specifically for (I) cell identification and tracking of cells in tissues, (2) tracking cell proliferation through multiple generations in tissues (3) vasculature location, and (4) deep 3-D imaging of Qdot-labeled polymer scaffold structures relative to the Qdot-labeled cells in engineered tissue models. Quantum Dots Corporation will prepare new types of Qdots that have properties suited to deep imaging of cells and structures in tissues. Particular emphasis will be placed on near infrared Qdots for imaging deeper in tissues. The Science and Technology Center at Carnegie Mellon University will then develop methods to derivatize Qdots for existing and new applications in cell biology. The STC will initially use available Qdots and conjugates, label cells by a variety of means, then test the cells for fluorescent brightness, stability of labeling, and cell survival and function. As newer quantum dots become available, they will be similarly tested and compared with existing Qdots and existing organic fluorescent probes, e.g., Alexa dyes and cyanine dyes. Fluorescence lifetime imaging capability will be added to the 3-D grating imager in the STC for enhancing signal-to-background in deep tissue imaging by taking advantage of the relatively long emission lifetime of Qdots. To obtain feedback for the development program we have included collaboration with the recently formed Bone Tissue Engineering Center at Carnegie Mellon University. In this collaboration we will examine the utility of the developing Qdot technology for studying cell location, movement and proliferation in the 3-D structures of engineered bone tissue. This is a particularly challenging and relevant system that requires Qdot technology to extend cell tracking to denser and more highly scattering tissue matrices, including hydroxyapatite-containing artificial bone matrices. By the conclusion of this project, we expect to have instrumentation and probes to perform time-resolved multicolor imaging at millimeter depth in many natural and artificial tissues. Thus the technology will be generic and have utility in many biological and medical applications.